State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing 210029, China; Department of General Surgery, Yixing Traditional Chinese Medicine Hospital, Wuxi 214200, China.
J Pediatr Surg. 2013 Oct;48(10):2099-105. doi: 10.1016/j.jpedsurg.2013.07.011.
BACKGROUND/PURPOSE: Hirschsprung's disease (HSCR) is a congenital disorder characterized by the absence of intramural ganglion cells which are highly associated with impaired proliferation and migration of neural crest cells. Whether methyl CpG binding protein 2 (MeCP2) is related with HSCR still remains unknown. This study investigates the involvement of MeCP2 in HSCR.
Quantitative real time PCR and western blot were used to detect the expression level of MeCP2 both in the aganglionic/diseased segment and the ganglionic/normal segment. In vitro assays we used siRNAs to knock-down the expression of MeCP2 in SH-SY5Y cell lines, and furthermore, MTT and transwell assays were used to detect the proliferation and migration ability, respectively. In addition, bisulfite sequencing (BSP) and miRNA analysis were used to examine why MeCP2 is decreased in HSCR samples.
MeCP2 exhibited a lower expression level in tissues of HSCR patients compared with the controls. The down-regulation may also suppress the proliferative ability of the cells. However, there was no significant difference in the MeCP2 methylation level between cases and controls. Similarly, there was no difference between cases and controls in miRNA-34b (miR-34b) which is predicted to regulate MeCP2 through complementary binding to the 3'-untranslated region of MeCP2.
Our results indicated that an aberrant decreased level of MeCP2 may play an important role in the pathogenesis of HSCR.
背景/目的：先天性巨结肠（HSCR）是一种先天性疾病，其特征是固有神经节细胞缺失，而神经嵴细胞的增殖和迁移受损与此高度相关。甲基化 CpG 结合蛋白 2（MeCP2）是否与 HSCR 有关尚不清楚。本研究探讨了 MeCP2 在 HSCR 中的作用。
采用定量实时 PCR 和 Western blot 检测无神经节/病变段和有神经节/正常段 MeCP2 的表达水平。在体外实验中，我们使用 siRNA 敲低 SH-SY5Y 细胞系中的 MeCP2 表达，进一步通过 MTT 和 Transwell 实验检测细胞增殖和迁移能力。此外，亚硫酸氢盐测序（BSP）和 miRNA 分析用于检测 HSCR 样本中 MeCP2 减少的原因。
与对照组相比，HSCR 患者组织中 MeCP2 的表达水平较低。这种下调可能也抑制了细胞的增殖能力。然而，病例组和对照组之间 MeCP2 的甲基化水平没有差异。同样，病例组和对照组之间 miRNA-34b（miR-34b）也没有差异，miR-34b 被预测通过与 MeCP2 的 3'非翻译区互补结合来调节 MeCP2。
我们的结果表明，MeCP2 的异常下调可能在 HSCR 的发病机制中起重要作用。
